Pharmaceutical, cosmetic and/or food composition with antioxidant properties

ABSTRACT

A pharmaceutical, cosmetic, and/or food composition containing a pyrazine derivative useful for preparing a medicament for preventing and/or treating pathologies related to the activity of oxygen promoters or for treating cancer tumors. The pathologies which can be treated include inflammatory diseases, carcinogenic diseases, atherosclerosis, or cancerous tumors. The pyrazine may be coelenteramine or its derivatives.

This is the U.S. national phase under 35 U.S.C. §371 of InternationalApplication PCT/BE98/00044, filed Mar. 30, 1998.

OBJECT OF THE INVENTION

The present invention relates to a pharmaceutical, cosmetic and/orfoodstuff composition intended in particular for the prevention and/ortreatment of diseases associated with pro-oxidizing agents.

The present invention also relates to the use of the pharmaceutical,cosmetic and/or foodstuff composition according to the invention.

TECHNOLOGICAL BACKGROUND AND PRIOR ART

Patent Application WO96/28160 describes a pharmaceutical, cosmeticand/or foodstuff composition having antioxidizing properties andcomprising a pyrazine derivative or a precursor thereof, the derivativesbeing imidazolopyrazines, such as coelenterazine.

This document also describes the use of such a pharmaceutical, cosmeticand/or foodstuff composition for treatment of diseases associated withthe action of pro-oxidizing agents, such as inflammatory diseases, orthe use of the said composition for treatment of cancerous tumours.

Antioxidizing molecules, such as vitamins, such as vitamin E (soluble inlipids), or cysteine derivatives (soluble in water) have also alreadybeen used in cosmetic, pharmaceutical and/or foodstuff applications.However, such antioxidizing molecules are characterized either by a lowsolubility in water or by a high toxicity, by too low an efficacy or bya low stability with respect to oxygen.

OBJECT OF THE INVENTION

The object of the present invention is to obtain a pharmaceutical,cosmetic and/or foodstuff composition which allows bonding ofpro-oxidizing agents and should not have the disadvantages of the priorart.

A particular object of the present invention is to obtain apharmaceutical, cosmetic and/or foodstuff composition advantageouslyallowing the prevention and/or treatment of diseases associated withpro-oxidizing agents.

Another object of the present invention is to provide a pharmaceutical,cosmetic and/or foodstuff composition which is characterized by animproved stability with respect to products of the prior art, by a lowtoxicity or an absence of toxicity, and by a significant solubility in alarge number of solvents and/or lipids.

Characteristic Elements of the Present Invention

The present invention relates to a pharmaceutical, cosmetic and/orfoodstuff composition comprising a pyrazine derivative of the formula

in which the radicals R¹ to R⁹ are H, radicals chosen from the groupconsisting of alkyl, alkenyl, alkinyl, aryl, arylalkyl, alkylaryl,heteroaryl, heteroalkyl and hetero-(alkylaryl and arylalkyl), preferablyhaving 1 to 20 carbon atoms and optionally containing 1 to 10heteroatoms, the carbon atoms of which can optionally be substituted byany element of the Mendeleev table, preferably an element chosen fromthe group consisting of H, B, N, O, F, P, S, Cl, As, Se, Br, Te and I,or chains of the formula (R⁵×R⁶)_(n), where n≧1, x represents one ormore heteroatoms and R⁵ and R⁶ are radicals chosen from the groupconsisting of alkyl, alkenyl, alkinyl, aryl, arylalkyl, alkylaryl,heteroaryl, heteroalkyl and hetero-(alkylaryl and arylalkyl) having 1 to20 carbon atoms and optionally containing 1 to 10 heteroatoms, thecarbon atoms of which can optionally be substituted by any element ofthe Mendeleev table, preferably an element chosen from the groupconsisting of H, B, N, O, F, P, S, Cl, As, Se, Br, Te and I; andoptionally a suitable pharmaceutical, cosmetic and/or foodstuff vehicle.

Preferably, in the pyrazine derivative according to the invention, theradicals R² to R⁹ are H and the radical R¹ is a radical of the formula

and optionally a suitable pharmaceutical, cosmetic and/or foodstuffvehicle.

Another aspect of the present invention relates to the use of thecomposition according to the invention for bonding pro-oxidizing agents(activated forms of oxygen), such as peroxides, superoxides etc.

The present invention also relates to a process for bondingpro-oxidizing agents in which the pharmaceutical, cosmetic and/orfoodstuff composition is brought into contact with the saidpro-oxidizing agents.

The present invention also relates to a process for treatment and/orprevention of diseases associated with the action of pro-oxidizingagents, in particular inflammatory diseases, and a process for treatmentand/or prevention of atherosclerosis, in which the pharmaceutical,cosmetic and/or foodstuff composition according to the present inventionis administered to a patient (human or animal).

In the said processes, the pharmaceutical, cosmetic and/or foodstuffcomposition is combined with a therapeutic effect. However, the saidcomposition can also be characterized as being a “functional” foodstuffcomposition.

A “functional foodstuff composition” is characterized in that itcomprises one or more ingredients which are capable of having abeneficial physiological effect on the consumer, such as the preventionof an illness, the treatment of an illness or the activation ofbiorhythm or the immune system. Such a functional foodstuff compositioncan be incorporated into the normal diet of the consumer such that ageneral improvement in the health of the patient is obtained, or toobtain treatment or prevention of a particular illness, in as much asthe said functional foodstuff composition has a significant effect onthe consumer from both the preventive and the therapeutic point of view.

The said pharmaceutical, cosmetic and/or foodstuff composition willcomprise a sufficient amount of the pyrazine derivative according to theinvention to obtain a prevention or a significant therapeutic effect onthe consumer.

The proportion of pyrazine derivative according to the invention in thepharmaceutical, cosmetic and/or foodstuff composition according to theinvention will vary as a function of the daily amounts of the productabsorbed, in accordance with suitable foodstuffs, cosmetics andpharmaceuticals legislation, sensory considerations and the secondaryeffects which may exist with the derivatives according to the inventionand/or their pharmaceutical, cosmetic and/or foodstuff vehicles used.

The pharmaceutical, cosmetic and/or foodstuff vehicles of thecompositions according to the invention are suitable vehicles inparticular for oral administration, for example in the form of coated ornon-coated tablets, pills, capsules, solutions, essential oils and/orsyrups.

Other suitable pharmaceutical, cosmetic and/or foodstuff vehicles can beused, depending on the mode of administration chosen.

In particular, these pharmaceutical, cosmetic and/or foodstuff vehiclescan be sun creams or oils well-known to the person skilled in the art,which may be smeared over various areas of the human or animal body incombination with other skin-protection agents.

In addition, the products of the invention can easily be incorporatedinto solvents (aqueous media, alcohols etc.) or lipids (for example incombination with foodstuff oils or sun-tanning oils).

The pharmaceutical, cosmetic and/or foodstuff compositions according tothe invention are prepared by processes generally used by the personskilled in the art, in particular by pharmacists, and can comprise anypharmaceutically suitable, solid or liquid non-toxic vehicle oradditive.

Incorporation of the derivatives according to the invention into agalenical medium may also be envisaged.

The percentage of active product (pyrazine derivative) in thepharmaceutical, cosmetic and/or foodstuff vehicle can vary within verywide ranges, which are generally limited by the tolerance and the levelof acceptance of the composition by the consumer.

The limits are generally determined by the frequency of consumption ofthe composition by the consumer.

A last aspect of the present invention relates to the use of thecomposition according to the invention for the preparation of amedicament intended for prevention and/or treatment of diseasesassociated with the action of pro-oxidizing agents.

In particular, the present invention relates to the use of thecomposition according to the invention for the preparation of amedicament intended for prevention and/or treatment of inflammatory orcarcinogenic diseases, prevention of atherosclerosis and/or treatment ofcancerous tumours.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the percentage cell survival of rat hepatocytes as afunction of increasing (molar) doses of coelenteramine.

FIG. 2 shows the oxidation of “low-density lipoproteins” (LDL) with afree-radical water-soluble initiator (AAPH) in the presence ofcoelenteramine.

FIG. 3 shows the course of the oxidation of human “low-densitylipoproteins” by Cu²⁺ as a function of time.

FIG. 4 illustrates the percentage survival of hepatocytes subjected tothe action of nitrofurantoin.

FIG. 5 illustrates the increase in the resistance of fibroblastssubjected to the action of an oxidative stress (increasing concentrationof t-BHP) in the presence or in the absence of coelenteramine.

FIG. 6 illustrates the reduction in the release of lactate dehydrogenaseby rat hepatocytes when increasing doses of coelenteramine are added tothe culture medium.

EXAMPLES

Coelenteramine (CLM) is a naturally occurring product present in marineorganisms which is derived from the oxidation of coelenterazine.

The structure of coelenteramine is illustrated below. This structureshows the following characteristics:

Example 1

Stability

Coelenteramine is characterized by an excellent stability towardsoxygen, both in the form of a powder and after solubilization in aqueousand organic solvents. In fact, this molecule is not changed by remainingin an aqueous solvent exposed to air for several days. After severaldays, no degradation product of coelenteramine was identified by HPLCand TLC chromatographic analysis.

Example 2

Toxicity

Coelenteramine is characterized by an absence of toxicity both onhepatic cells and on intestinal cells or fibroblasts. Coetenteramine wasapplied to primary cultures of rat hepatocytes cultured in microplates(20,000 cells per well, 200 μl ). The doses of CLM ranged from 1.3×10⁻⁵to 5×10⁻⁴ M. After a period of 24 hours, their survival was evaluated bymeasuring the contents of total proteins in each well (Bradford method).The results, shown on FIG. 1, expressed in percentage survival ofcontrols not treated with CLM, show that the latter is not at all toxicto the hepatocytes at these concentrations. Similar results wereobtained on human fibroblasts (MRC-5) and human intestinal cells(CACO2).

Example 3

Protective Activity of Lipoproteins

Atherosclerosis constitutes a major cause of cardiovascular mortality.Epidemiological studies have shown that an accelerated development ofatherosclerosis is associated with a high plasma level of “low-densitylipoproteins” (LDL). It is known that LDL contain various antioxidizingagents, the major element of which is α-tocopherol (vitamin E). Ascorbicacid (vitamin C), ubiquinone and β-carotene represent the other mainantioxidizing molecules of LDL. It has been demonstrated in vitro thatthe conversion of macrophages into spumous cells charged with lipids(which are the main constituents of the atherosclerosis plaque) isassociated with a change in LDL of an oxidative nature. The oxidation ofLDL is a process mediated by free radicals and initiated by peroxidationof polyunsaturated fatty acids after consumption of antioxidizingagents. The oxidation of LDL greatly increases atherogenic properties.Epidemiological studies have shown an inverse relationship betweencardiovascular mortality and the plasma concentration of antioxidants(such as vitamin E). Consequently, water- and liposoluble antioxidantscan protect LDL and delay or prevent their oxidation process. Thus, inorder to estimate the protective effect of coelenteramine againstatherosclerosis, the in vitro oxidation of LDL in the absence or in thepresence of varying concentrations of coelenteramine is compared.

Oxidation of the polyunsaturated fatty acids of LDL is accompanied bythe formation of joint dienes, which have a maximum absorbance at 234nm. These joint dienes result from rearrangement of the double bonds ofpolyunsaturated fatty acids following removal of a malonic hydrogen. Theincrease in the absorption of joint dienes can be measured directly insolution without resorting to extraction of the lipids. A latency phaseduring which few dienes are produced, a propagation phase during whichthe absorbance at 234 nm increases rapidly to reach a plateau and adecomposition phase where the aldehydes formed also absorb in the regionof 210-240 nm, thus producing a new increase in the absorbance at 234nm, are observed in succession. Measurement of the conjugated dienescurrently seems to be the best indicator of the oxidizability of LDL ifpure LDL preparations are available (Esterbauer et al. (1989)).

In an acellular medium, oxidation of LDL can be catalysed by metal ions,such as copper, or induced by a water-soluble free-radical initiator,such as 2,2′-azobis(2-amidinopropane) (AAPH).

To measure the joint dienes, a solution of LDL (final concentration of50 μg protein/ml PBS buffer, pH 7.4) is incubated in the presence of 5μM copper or 2 μM AAPH and in the absence or in the presence of varyingconcentrations of CLM in a quartz cell with an optical path of 1 cm. Theabsorbances at 234 nm of various samples are measured as a function oftime on a UV-visible spectrophotometer (DU-8, Beckman). A blank isobtained by means of a phosphate solution without LDL but containing 5μM CuCl₂.2H₂O and 2.5 μM CLM. The tests are carried at 30° C. foroxidation of the LDL in the presence of copper and at 37° C. in thepresence of AAPH.

The results indicate that CLM has a significant protective action on theLDL with respect to their oxidation by AAPH or copper. The action of CLMon the oxidation of LDL by AAPH is illustrated in FIG. 2, in which thesignificant slowing down in the appearance of joint dienes withincreasing concentrations of CLM is seen. Similar results are found ifthe oxidation of the LDL is realized with copper. FIG. 3 illustrates thedelay in the appearance of TBARS (thiobarbituric acid reactivesubstances), essentially corresponding to malonaldialdehyde (MDA), aproduct of the oxidation of the lipids contained in LDL.

Example 4

Protective Action on Cells Subjected to an Oxidative Stress

Primary cultures of hepatocytes were subjected to the action oftert-butyl hydroperoxide (t-BHP) and nitrofurantoin (NF). The action oft-BHF is associated with its direct oxidizing power, while the action ofNF results from the production of superoxide anions during itsmetabolization by the cytochrome P450 of cells.

FIG. 4 illustrates the survival of hepatocytes subjected to the actionof 3×10⁻⁴ M NF for 6 hours. At the end of this treatment, the survival(estimated by the MTT method) was 18% in the control groups. If CLM isadded to the culture medium, the survival of the hepatocytes increasesregularly, to reach 70% of the controls at 5×10⁻⁵ M CLM.

FIG. 5 illustrates the increase in the resistance of fibroblastssubjected to the action of t-BHP by means of 10⁻⁵ M CLM. In this case, asignificant displacement of the cytotoxicity curve (measured by the MTTtest) of t-BHP towards higher concentrations in the presence of CLM isobserved.

Similar results were observed in hepatocytes treated with t-BHP. FIG. 6illustrates the reduction in the release of lactate dehydrogenase (LDH),a cytotoxicity indicator, by cells if CLM (10 and 50 μM) is added to theculture medium. The mortality, which is 75% in the controls, reachesonly a few % in the presence of 50 μM CLM.

What is claimed is:
 1. A pharmaceutical, cosmetic and/or foodstuffcomposition comprising in a carrier selected from the group consistingof pharmaceutical, cosmetic, and or foodstuff a pyrazine derivative ofthe formula

wherein the radicals R¹ to R⁹ are selected from the group consisting ofH, radicals or substituted radicals selected from the group consistingof alkyl, alkenyl, alkinyl, aryl, arylalkyl, alkylaryl, heteroaryl,heteroalkyl, and hetero-(alkylaryl and arylalkyl), and chains of theformula (R⁵ x R⁶)_(n), where n≧1, x represents one or more heteroatoms,and R⁵ and R⁶ are radicals or substituted radicals selected from thegroup consisting of alkyl, alkenyl, alkinyl, aryl, arylalkyl, alkylaryl,heteroaryl, heteroalkyl and hetero-(alkylaryl and arylalkyl).
 2. Thepharmaceutical, cosmetic and/or foodstuff composition according to claim1, wherein the radicals R² to R⁹ are H, and the radical R¹ is a radicalof the formula


3. A method for bonding pro-oxidizing agents, comprising contacting thepharmaceutical, cosmetic and/or foodstuff composition according to claim1 with said pro-oxidizing agents.
 4. A method for prevention and/ortreatment of diseases associated with the action of pro-oxidizingagents, comprising: preparing a medicament comprising the pharmaceuticalcomposition according to claim 1; and administering an effective amountof said medicament to a subject in need of treatment.
 5. A method forprevention and/or treatment of inflammatory diseases comprising:preparing a medicament comprising the pharmaceutical compositionaccording to claim 1; and administering an effective amount of saidmedicament to a subject in need of treatment.
 6. A method for theprevention and/or treatment of carcinogenic diseases comprising:preparing a medicament comprising the pharmaceutical compositionaccording to claim 1; and administering an effective amount of saidmedicament to a subject in need of treatment.
 7. A method for preventionand/or treatment of atherosclerosis comprising: preparing a medicamentcomprising the pharmaceutical composition according to claim 1; andadministering an effective amount of said medicament to a subject inneed of treatment.
 8. A method for treatment of cancerous tumorscomprising: preparing a medicament comprising the pharmaceuticalcomposition according to claim 1; and administering an effective amountof said medicament to a subject in need of treatment.
 9. A method forbonding pro-oxidizing agents, comprising contacting the pharmaceutical,cosmetic and/or foodstuff composition according to claim 2 with saidpro-oxidizing agents.
 10. A method for prevention and/or treatment ofdiseases associated with the action of pro-oxidizing agents, comprising:preparing a medicament comprising the pharmaceutical compositionaccording to claim 2; and administering an effective amount of saidmedicament to a subject in need of treatment.
 11. A method forprevention and/or treatment of inflammatory diseases comprising:preparing a medicament comprising the pharmaceutical compositionaccording to claim 2; and administering an effective amount of saidmedicament to a subject in need of treatment.
 12. A method for theprevention and/or treatment of carcinogenic diseases comprising:preparing a medicament comprising the pharmaceutical compositionaccording to claim 2; and administering an effective amount of saidmedicament to a subject in need of treatment.
 13. A method forprevention and/or treatment of atherosclerosis comprising: preparing amedicament comprising the pharmaceutical composition according to claim2; and administering an effective amount of said medicament to a subjectin need of treatment.
 14. A method for treatment of cancerous tumorscomprising: preparing a medicament comprising the pharmaceuticalcomposition according to claim 2; and administering an effective amountof said medicament to a subject in need of treatment.